The invention relates to methods and compositions for diagnosing and treating conditions requiring regulation of trophoblast invasion.
During placental development the establishment of fetal-maternal interactions is critical for a successful human pregnancy (1). Abnormalities of placenta formation due to shallow trophoblast invasion have been linked to preeclampsia and fetal growth restriction (2). In contrast, uncontrolled trophoblast invasion and abnormal trophoblast growth are associated with hydatiform mole and choriocarcinoma. In the course of placenta formation, chorionic villous cytotrophoblasts undergo two morphologically distinct pathways of differentiation. The vast majority of cytotrophoblasts in both floating and anchoring villi fuse to form the syncytiotrophoblast layer, which permits gas and nutrient exchange for the developing embryo. A small percentage of cytotrophoblasts in anchoring villi break through the syncytium, at selected sites, and generate columns of non-polarized cells which migrate into the endometrium. These extravillous trophoblasts (EVT) invade deeply into the uterus reaching the first third of the myometrium at which point they invade the spiral arteries, replacing their endothelium and vascular wall. Invasion peaks at 12 weeks of gestation and rapidly declines thereafter. indicating that, unlike tumour invasion, it is spatially and temporally regulated (3). Trophoblast invasion in the decidua is accompanied by a complex modulation of the synthesis and degradation of extracellular matrix (ECM) proteins and in the expression of adhesion molecules (4-6). Along the invasive pathway, ECM proteins undergo changes in their spatial distribution with loss of laminin and appearance of fibronectin (3,4). EVT loose the expression of E-cadherins, responsible for cell-cell adhesion between polarized stem cytotrophoblasts, down-regulate xcex16 xcex24 integrin, a laminin receptor, and acquire xcex15xcex21 integrin, a fibronectin recepor (7). Once the EVT invade the endometrium they express the xcex11xcex21 integrin, a collagen/laminin receptor. Thus, specific changes in ECM proteins and their receptors are associated with the acquisition of an invasive phenotype by the extravillous trophoblasts (4).
Preeclampsia occurs in 5-10% of pregnancies and is the leading cause of death and illness in women during pregnancy. Preeclamnpsia is also associated with considerable fetal/neonatal complications because of adverse intrauterine conditions and preterm delivery. There is currently no effective pharmacologic treatment for preeclampsia and the only remedy is to remove the placenta (and hence deliver the fetus preterm). Current protocols, including bedrest and antihypertensive drugs, seek to stabilize maternal/fetal condition until delivery is necessitated. It is estimated that around 200,000 children are born preterm in North America due to preeclampsia. Many of these babies will require costly intensive care at birth and if they survive may face a lifetime of chronic illness (e.g. lung disease) or disability (e.g. cerebral palsy, mental handicaps, blindness). These conditions represent a significant impact on subsequent requirements for community health care resources. Therefore, reducing the incidence of preeclampsia and preterm birth would have a tremendous positive impact on health care delivery.
The present inventors have studied the mechanisms that regulate trophoblast invasion. The inventors have found that antisense disruption of the expression of the TGFxcex2 receptor, endoglin, triggers invasion of cytotrophoblast from first trimester villous explants in vitro indicating that the TGFxcex2 receptor system, and in particular endoglin, plays a critical role in regulating this process. Significantly, the present inventors defined components that endogenously regulate trophoblast invasion. TGF-xcex23 was found to be a major regulator of trophoblast invasion in vitro. In particular, the presence of TGF-xcex23 and its receptors at 5-8 weeks at a time when there is no spontaneous trophoblast invasion and the absence of these molecules at 12-13 weeks when spontaneous invasion occurs, establishes a major role for TGF-xcex23 as an endogenous inhibitor of trophoblast invasion. Down-regulation of TGF-xcex23 (but not xcex21 or xcex22) expression using antisense oligonucleotides, stimulated extravillous trophoblast cell (EVT) outgrowth/migration and fibronectin production in 5-8 villous explants indicating that TGF-xcex23 acts to suppress in vivo trophoblast invasion. The effects of antisense treatment to TGF-xcex23 are specific as they are prevented by addition of exogenous TGF-xcex23 but not TGF-xcex21 or TGF-xcex22. The stimulatory effects of TGF-xcex23 are lost after 9 weeks of gestation which is compatible with TGF-xcex23 being produced by the villi during a specific window of gestation within the first trimester (5-8 weeks) and that inhibition of its synthesis stimulates trophoblast differentiation. Addition of exogenous TGF-xcex23 to the villous explants inhibits fibronectin synthesis.
The clinical importance of TGF-xcex23 in regulating trophoblast invasion has been highlighted by the finding that TGF-xcex23 is highly expressed in trophoblast tissue of preeclamptic patients when compared to that in age-matched control placenta while there was no change in the expression of either the xcex21 or xcex22 isoform. Fibronectin and xcex15 integrin expression were also greater in preeclamptic placenta, indicating that in preeclampsia, where there is shallow trophoblast invasion, trophoblast cells are arrested as an xcex15 integrin phenotype producing TGF-xcex23. These data are supported by the finding that villous explants from a control (non-preeclamptic placenta, 32 weeks of gestation) spontaneously formed columns of trophoblasts that invaded the surrounding Matrigel, while explants from a preeclamptic placenta did not.
In contrast to TGF-xcex23, activin, a TGF-xcex2 receptor, has been found to trigger trophoblast invasion. Follistatin an activin binding protein, inhibited the stimulatory effect of activin, and antibodies and antisense to endoglin.
Oxygen tension was also found to play a role in regulating trophoblast invasion. The expression of the hypoxia inducible factor, HIF-1xcex1, parallels that of TGF-xcex23 in first trimester trophoblast (i.e. peaks at 6-8 weeks but decreases after 9-10 weeks when oxygen tension increases). Expression of HIF-1xcex1 was dramatically increased in placentas of preeclamptic patients when compared to age-matched control tissue. Induction of HIF-1xcex1 by low PO2 (around 6-8 weeks) up regulates TGF-xcex23 transcription and blocks trophoblast invasion. A failure of the system to down-regulate at 9-11 weeks (either due to a block in response to normoxia or the absence of an increase in oxygen tension) leads to shallow invasion and predisposes to preeclampsia.
In addition to endoglin, the present inventors have found that TGF-xcex23 signals through a receptor complex which includes RI (ALK1), RII and endoglin. While TGF-xcex2 RI (ALK-5) and TGF-xcex2 R-II are expressed throughout the villi and decidua at 9-10 weeks gestation, they were found to be absent from the base of the proximal columns of the anchoring villi at the transition zone between the villous and the invading EVT exactly at the site where endoglin is up-regulated. This dramatic change in TGF-xcex2 receptor expression indicates that EVT within the columns in situ are not subject to the inhibitory actions of TGFxcex2, but via R-I and R-II they come under the control of this ligand upon entering the decidua. In addition, antisense induced disruption of RI (ALK-1) and RII expression stimulated trophoblast outgrowth/migration and fibronectin synthesis. In contrast, antisense to RI (ALK-5) inhibited fibronectin synthesis.
Broadly stated the present invention relates to a method for detecting, preventing, and/or treating a condition requiring regulation of trophoblast invasion by modulating (a) TGFxcex23 (b) receptors of cytokines of the TGFxcex2 family, (c) HIF-1xcex1, and/or (d) O2 tension. In accordance with one aspect of the invention a method is provided for diagnosing in a subject a condition requiring regulation of trophoblast invasion comprising detecting TGFxcex23, receptors of cytokines of the TGFxcex2 family, or HIF-1xcex1, in a sample from the subject. In an embodiment of the diagnostic method of the invention, a method is provided for diagnosing increased risk of preeclampsia in a subject comprising detecting TGFxcex23 or its receptors, or HIF-1xcex1 in a sample from the subject.
The invention also broadly contemplates a method for regulating trophoblast invasion comprising inhibiting or stimulating TGFxcex23, receptors of cytokines of the TGFxcex2 family. HIF-1xcex1 or O2 tension. In an embodiment of the invention, a method is provided for increasing trophoblast invasion in a subject comprising administering to the subject an effective amount of an inhibitor of (a) TGFxcex23, (b) receptors of cytokines of the TGFxcex2 family, and/or (c) HIF-1xcex1. In a preferred embodiment of the invention a method is provided for treating a woman suffering from, or who may be susceptible to preeclampsia comprising administering therapeutically effective dosages of an inhibitor of (a) TGFxcex23, (b) receptors of cytokines of the TGFxcex2 family, and/or (c) HIF-1xcex1. A therapeutically effective dosage is an amount of an inhibitor of (a), (b) and/or (c) effective to down regulate or inhibit TGFxcex23 in the woman.
In another embodiment of the invention, a method is providing for reducing trophoblast invasion in a subject comprising administering an effective amount of (a) TGFxcex23; (b) receptors of cytokines of the TGFxcex2 family; (c) HIF-1xcex1; and/or (d) a stimulator of (a), (b) or (c). In a preferred embodiment, a method is provided for monitoring or treating choriocarcinoma or hydatiform mole in a subject comprising administering therapeutically effective dosages of (a) TGFxcex23; (b) receptors of cytokines of the TGFxcex2 family; (c) HIF-1xcex1; and/or (d) a stimulator of (a), (b) or (c). An amount is administered which is effective to up regulate or stimulate TGFxcex23 in the subject.
The invention also relates to a composition adapted for regulating trophoblast invasion comprising a substance which inhibits or stimulates TGFxcex23 receptors of cytokines of the TGFxcex2 family, and/or HIF-1xcex1, or regulates O2 tension, in an amount effective to inhibit or stimulate trophoblast invasion, and an appropriate carrier, diluent, or excipient. In an embodiment of the invention, a composition is provided for treating a woman suffering from, or who may be susceptible to preeclampsia, comprising a therapeutically effective amount of an inhibitor of (a) TGFxcex23, (b) receptors of cytokines of the TGFxcex2 family, and/or (c) HIF-1xcex1, and a carrier, diluent, or excipient. In another embodiment of the invention, a composition is provided for monitoring or treating choriocarcinoma or hydatiform mole in a subject comprising a therapeutically effective amount of (a) TGFxcex23; (b) receptors of cytokines of the TGFxcex2 family; (c) HIF-1xcex1; and/or (d) a stimulator of (a), (b) or (c), and a carrier, diluent, or excipient.
The invention further relates to a method of selecting a substance that regulates trophoblast invasion comprising assaying for a substance that inhibits or stimulates TGFxcex23, receptors of a cytokine of the TGFxcex2 family, or HIF-1xcex1. The substances may be used in the methods of the invention to regulate trophoblast invasion.
The invention also relates to kits for carrying out the methods of the invention.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.